Transferrin (hereinafter sometimes referred to as “TF”) is one of glycoproteins which bind to Fe3+ ions and is deeply involved in iron metabolism. Particularly human serum transferrin (hereinafter sometimes referred to as “human transferrin” or “hTF”) belongs to a family of iron-binding proteins in a human body, is involved in iron transport/metabolism in the body and is used as additives to be used for a medium for animal cell culture, a pharmaceutical preparation or a DDS carrier. TF is an about 80 kDa glycoprotein having two domains named N-lobe and C-lobe, and it is synthesized by translation as an immature protein having a secretion signal peptide at its N-terminal and is then secreted out of the cell as a mature glycoprotein having the secretion signal peptide cleaved.
As the most common method for TF production, a method for producing a full-length recombinant protein by using a cell line of mammal cells such as baby hamster kidney cells (BHK cells) has been known. Further, a method of using budding yeast S. pombe has been known. S. pombe has, as different from budding yeast Saccharomyces cerevisiae, cell division and transcription forms close to those of human cells and contains no substance which has adverse effects on the body. Accordingly, the method for producing TF full-length recombinant protein employing S. pombe is excellent as a method for producing TF to be administered to human, such as a pharmaceutical preparation. For example, Patent Document 1 discloses a method such that a transformant using S. pombe as a host and having a hTF gene introduced is cultured in a liquid medium containing casamino acids to efficiently produce hTF, which is made to be secreted to the liquid medium and recovered.
On the other hand, in a heterologous protein production system employing an eukaryotic microorganism such as a yeast, in order to improve the efficiency for production of the desired heterologous protein, it has been known to use an improved host having a part of or the entire genome portion of a host unnecessary or disadvantageous for heterologous protein production eliminated or inactivated. For example, Patent Document 2 discloses that use of an improved host having at least one gene selected from genes encoding a specific protease (protease gene group) of S. pombe eliminated or inactivated, improves the efficiency for production of a heterologous protein.
Further, hTF protein can be secreted and produced by production in a state where a secretion signal (endoplasmic reticulum targeting signal) peptide is added to the N-terminal of hTF protein. For example, by culturing a transformant of S. pombe which has introduced a structural gene encoding a fusion protein having a secretion signal peptide recognized by S. pombe, such as a polypeptide derived from secretion signal of a precursor of a mating pheromone (P-factor) relating to mating of S. pombe, fused to the N-terminal of hTF protein, from the produced fusion protein, secretion signal peptide is removed in the Golgi apparatus or the endoplasmic reticulum, and hTF protein is secreted into the medium. For example, Patent Document 3 discloses that by expressing, at the N-terminal side of the desired foreign protein, a partial protein composed of a secretion signal peptide of PDI1 (protein disulfide isomerase 1) which is a protein having a molecular chaperone function and localized in the endoplasmic reticulum, an a-domain, a b-domain and an x-domain, or a partial protein composed of an endoplasmic reticulum targeting signal peptide of PDI1, an a-domain, a b-domain, a b′-domain and an x-domain, in a fused state, the amount of secretory production of the foreign protein by S. pombe can be increased. PDI1 comprises, from the N-terminal, an ER targeting signal, a-domain, b-domain, b′-domain, x-domain, a′-domain and c-domain containing an ER retention signal (ADEL) (SEQ ID NO: 23), in this order. Each of the a-domain and the a′-domain has one active site (CGHC) (SEQ ID NO: 24) for molecular chaperone activity. All the four domains a, b, b′ and a′ form a thioredoxin fold.